The combination of raloxifene and epigallocatechin gallate suppresses growth and induces apoptosis in MDA-MB-231 cells

Previous studies have demonstrated that raloxifene induces apoptosis in a variety of cancer cell lines. We aimed to determine if this effect was enhanced by combining raloxifene with epigallocatechin gallate (EGCG). Results demonstrated that EGCG (25 mu M) and raloxifene (1-5 mu M) produced enhanced cytotoxicity toward MDA-MB-231 breast cancer cells compared to either drug alone following 7 days of treatment. The combination of 5 pM raloxifene and EGCG was the most effective as it decreased cell number by 96% of control, and time-course studies demonstrated that significant cytotoxicity began 36 h after treatment. Potential mechanisms for this effect were then investigated. Flow cytometry experiments demonstrated that apoptosis was significantly increased following 12 h of combination treatment compared to all other treatment groups. A maximal increase in the proportion of cells in the G(1)-phase of the cell cycle (116% of control) occurred following 24 h of combination treatment, 12 h after the significant increase in apoptosis, and thus was not considered to be a viable mechanism for the enhancement of apoptosis. While raloxifene was a competitive inhibitor of microsomal UDP-glucuronosyltransferase activity (K-i of 24 pM), it did not decrease the metabolism of EGCG as the rate of disappearance of EGCG from the media was the same for cells treated with either EGCG or EGCG + raloxifene. Finally, the combination treatment reduced the phosphorylation of EGFR and AI(T proteins by 21.2 +/- 3.3% and 31.5 +/- 1.7% from control, respectively. In conclusion, the synergistic cytotoxicity elicited by the combination of EGCG and raloxifene results from an earlier and greater induction of apoptosis. This is likely to be a result of reduced phosphorylation of EGFR and AKT signaling proteins. (c) 2008 Elsevier Inc. All rights reserved.